Rotaviruses are the most important cause of viral gastroenteritis in humans and animals. the rotavirus genome consists of 11 segments of double- stranded (ds) RNA of 667 (segment 11) to 3,302 bp (segment 1). The general features of rotavirus transcription and replication probably are similar to those described for reovirus. Virions possess a viral polymerase which is contained together with the dsRNA segments in a subviral single-shelled core and directs copying of the parental minus strand into progeny plus strands (transcription). The plus strands serve as mRNAs and also as templates for the synthesis of progeny minus strands to yield dsRNA (replication). During the synthesis of the negative strand the nascent dsRNA associates with viral proteins to form subviral cores which eventually are incorporated into virions. The 5' and 3' ends of rotavirus RNAs contain distinct, unrelated terminal consensus sequences of 7 to 10nt which are assumed to be important cis- acting signals. These presumably include, at the 3' ends, the viral promoters for the synthesis of plus and minus strands. The termini also might contain sequences important for packaging and for regulation of expression at the levels of transcription, replication and translation. The 5' end of the plus strand has a methylated cap structure and neither strand is polyadenylated. Recently, it was shown by others that authentic plus-sense reovirus RNAs synthesized in vitro by subviral cores were infectious when transfected into cells and complemented with a helper reovirus from a different serotype. To date, however, similar results have not been reported with synthetic reovirus RNAs encoded by cDNA or with any rotavirus. The capability of introducing synthetic RNAs into infectious virus would provide the basis for performing detailed structure-function studies of the viral RNAs and proteins and for developing methods for engineering attenuated vaccine strains. Our approach has been to develop a simplified system in which cDNA-encoded synthetic analogs of a rotavirus RNA, consisting of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene flanked by the 5' and 3' gene 9 noncoding regions, were rendered biologically active by transfection into rotavirus-infected cells. This provides a new, sensitive system for characterizing rotavirus gene replication and expression.